CN102866135A - Time resolution fluorescent biosensor based on phosphorescent light emitting technology and application thereof - Google Patents

Abstract

The invention provides a time-resolved fluorescent biosensor based on phosphorescence technology and its application, including: an excitation optical path module, a photoelectric signal receiving and converting module, a control system module, an amplification and shaping circuit, a decoding interface circuit and a data buffer module. The excitation light path module includes a main detection excitation light module and an auxiliary optical scanning module; the photoelectric signal receiving conversion module includes a main detection photoelectric conversion module and an auxiliary scanning photoelectric conversion module. The invention adopts the phosphorescent luminescent material, that is, platinum/palladium porphyrin as the biomarker, and the result is expressed in the form of infrared light signal under the irradiation of green light, and can be interpreted by an instrument, so as to realize the quantitative detection of the target object to be detected. The present invention divides the chromatographic test strip into a sandwich method mode, a competition method mode, an indirect method mode and a capture method mode according to the different ways of the immunoreaction of the analytes to quickly and sensitively detect different analytes in the sample. Qualitative and quantitative detection analysis.

Description

Time-resolved fluorescent biosensor based on phosphorescence technology and its application

technical field

The invention relates to a time-resolved fluorescent biosensor based on phosphorescence technology and an application thereof, belonging to the technical field of immune detection.

Background technique

In order to solve the interference problem of FIA from samples, especially the strong and changing fluorescent background of biological samples, as well as scattering and other factors, a long-life, long-wavelength fluorescent label chromatography test strip (such as lanthanide metal chelate) has been greatly promoted. Compounds, porphyrin compounds) and the development of time-resolved fluorescence technology, the sensitivity of non-isotope labeling analysis has reached or exceeded the radioimmunoassay method. Phosphorescence analysis is the sister technology of fluorescence analysis. Compared with fluorescence, it has many unique advantages: (1) It has a large Stokes shift, the wavelength of phosphorescence is longer than that of fluorescence, and it is far away from the excitation spectrum , will not overlap with the excitation spectrum, not only can reduce or eliminate the interference of the background fluorescence of the sample (especially biological samples) and the incident excitation light, but also alleviate the self-absorption phenomenon; (2) due to T ₁ →S ₀ spin forbidden The lifetime of phosphorescence is longer than that of fluorescence, and the lifetime of phosphorescence is about 10 ^-3 ~ 10s, which is easy to realize time-resolved measurement; (3) the selectivity is better. But so far, phosphorescence immunoassay has not been developed as it should. The reason is mainly due to the limitation of phosphorescent materials, especially the lack of phosphorescent materials with high water solubility and good biocompatibility. Therefore, a phosphorescence technology that is suitable for immunoassay and can show the advantages of phosphorescence detection has not been developed. .

Metalloporphyrins are a class of macrocyclic compounds that widely exist in nature, such as heme, chlorophyll, VB12, etc. They play an important role in the metabolism of living organisms and many basic biological processes. The porphyrin molecule consists of four pyrrole rings linked by methine to form a four-coordinated porphyrin nucleus. The porphyrin ring is very stable and can coordinate with metals with a diameter of 3.7 angstroms; its complexes with transition metals are especially stable, such as Zn-tetraphenylporphyrin (ZnTPP), whose stability constant is 10 ²⁹ . Most metals form a 1:1 complex with porphyrin, only Na, K, and Li complexes have a ratio of 2:1, and the two metal atoms are located above and below the plane of the porphyrin ring, respectively. As shown in Figure 1, the principle of porphyrin energy transition to generate phosphorescence is described. The electronic absorption spectrum of porphyrin mainly has Soret band (also known as B band) and Q band. The Soret band is located between 400-450nm, and the molar absorptivity is high (2-5×10 ⁵ mol ^-1 .L.cm ^-1 ). The Soret band of metalloporphyrin has weak absorption, and when there are electrophilic groups on the side of the ring, the Soret band will move to the long-wave direction. The Q band of porphyrin is generally between 450 and 650 nm, and there are four related peaks; when the hydrogen on the pyrrole ring nitrogen is replaced by metal ions to form metalloporphyrin, the four related peaks weaken or disappear. Porphyrins and metalloporphyrins have 18-electron large π delocalized structures, so when their B-band or Q-band is used as the excitation wavelength, they all have different degrees of fluorescence emission between 600-700nm (or longer wavelength range); In general, the fluorescence intensity of metalloporphyrins is weaker than that of porphyrins. At room temperature, porphyrin itself does not emit phosphorescence, and it emits phosphorescence in the near-infrared region when it forms a complex with some metals and coexists with ordered media (such as surfactants, biomacromolecules such as proteins and nucleic acids); but only Very few metalloporphyrins phosphoresce, the most common being palladium porphyrins and platinum porphyrins. Palladium/platinum porphyrin has extremely strong phosphorescence, which is characterized by long lifetime (ms) and long wavelength (600-1000nm). The most common are water-soluble meso-tetrakis (4-sulfonic acid phenyl) porphyrin (H ₂ TSPP ^4- ) and meso-tetrakis (4-N-trimethylaminophenyl) porphyrin (H ₂ TMAP ⁴⁺ ) Palladium/platinum complexes, and palladium/platinum complexes of water-insoluble octaethylporphyrin (OEP) and tetraphenyl-tetrabenzoporphyrin (Ph ₄ TBP ), etc. The near-infrared region of 600-1000nm is an extremely useful region for studying bioluminescent probes and photochemical sensors. Therefore, palladium/platinum porphyrin with special phosphorescence characteristics has become a very effective probe molecule in biological analysis, which can provide high sensitivity and selectivity combined with some simple detection instruments.

As shown in accompanying drawing 4, under external excitation, platinum porphyrin emits strong phosphorescence at 650nm, duration 100 microseconds (absorption wave range 390-410nm), palladium porphyrin emits strong phosphorescence duration 500 microseconds at 670nm (absorption wave range 390-410nm). Wave range 400-420nm). These porphyrin particles also have a large Stokes shift (both 280nm). Compared with other luminescent materials, the advantage of platinum/palladium porphyrin lies in its minimal photobleaching, which can be effectively excited by using cheap and strong light sources, such as light-emitting diodes. In addition, the background fluorescence of biological samples and nitrocellulose membranes is lower when excited at 390-420 nm than at 365 nm for time-resolved fluorescence represented by europium ions. Although the transmission of 390-420nm light through the nitrocellulose membrane is not ideal, it is better than 365nm light and is more suitable for transmission measurement. Platinum porphyrin can also covalently label antibodies, providing a sensitive and rapid detection technique for the detection of various samples.

At present, the markers used in immunochromatography are usually enzymes, colloidal gold, and various colored microsphere markers. These markers have the same characteristics in immunochromatography: physical adsorption and color judgment Test results. Among them, the characteristics of physical adsorption labeling (that is, the principle of hydrophobicity and electrostatic adsorption) make it easy to form non-specific interference, and it is necessary to add non-specific interference elimination chromatography test strips to the production process formula, such as Tween 20 and other surfactants, etc. , but when using such chromatographic test strips, it is also easy to cause false positive or false negative results based on such markers. In addition, the results of color interpretation must be subject to the subjective influence of the observer when used, especially weak positive results, and can only make qualitative judgments, but cannot achieve accurate quantitative judgments. These shortcomings greatly limit the application of immunochromatography in clinical testing.

The publication number is CN102087293A, the publication date is June 8, 2011, the name is "an immunochromatographic test strip for the whole process of quantitative detection of troponin I and its preparation method" and the publication number is CN102087214, the publication date is 2011 On June 8, the patent application titled "Fluorescence Quantitative Detector" disclosed a chromatographic detection method based on fluorescent latex labeling. The fluorescence excitation wavelength is 470nm and the emission wavelength is 530nm. However, this kind of traditional organic fluorescent materials has not solved its inherent photobleaching problem; at the same time, the interference of the autofluorescence of biological samples and the photoinstability of organic fluorescent molecules also reduce the fluorescence signal of the analyte, which will inevitably lead to biased detection sensitivity. Low, the detection linear range is narrow, and it is difficult to meet the needs of clinical detection.

The patent application with the publication number CN102192983A, the publication date is September 21, 2011, and the title is "Time-resolved fluorescence immunochromatography quantitative detection test strip and its preparation method and application" discloses that it is filled with lanthanide rare earth elements Time-resolved fluorescent microspheres of and their chelates were used as labeled probes. But for the existing rare earth fluorescent biolabeling probes, a major disadvantage is that almost all lanthanide rare earth fluorescent probes need to be excited by ultraviolet light. So far, the known visible light-excited lanthanide rare earth element complexes cannot be used directly due to problems such as poor water solubility, instability in polar coordination solvents, low fluorescence quantum yield, or lack of active labeling groups. biomarker. This largely limits the application of such probes in the determination of living biological samples.

Chinese Patent No. ZL200410034104.0, titled "Immune Chromatography Test Strip Based on Up-Conversion Luminescence Technology" and Chinese Patent No. ZL200410034105.5, titled "Up-Conversion Luminescent Biosensor" both disclose an immune layer Analysis of test strips and detection methods. The commonly used up-conversion fluorescent materials are mainly based on fluoride and oxide, and doped with rare earth elements such as Yb and Er. The excitation light of the up-conversion nano-fluorescent material is infrared light, and biological samples have extremely low background fluorescence at this excitation wavelength, while the detection wavelength is in the visible region, and there is no problem of background fluorescence interference of complex matrix samples. The optical stability of the up-conversion nano-fluorescent material is good, and there is no photobleaching and fading phenomenon. At present, the main problems hindering the application of up-conversion nano-fluorescent materials in biochemical analysis are their low quantum yield and large particle size. Generally, it is difficult to obtain strong fluorescent up-conversion fluorescent materials with a particle size of less than 50nm.

The publication number is CN1811449, the publication date is August 2, 2006, and the title is "Detection method of quantum dot-labeled rapid immunochromatography test strip" and the publication number is CN101893623A, the publication date is November 24, 2010, and the title is The patent applications for "Ultrasensitive Quantum Dot Microsphere Immunochromatography Test Strip Rapid Detection Method" all disclose immunochromatography test strips based on quantum dot technology. Quantum dot nanoparticles can emit fluorescence under light excitation. As a new type of inorganic fluorescent nanomaterial, it has been widely used in the field of life sciences, and has shown good application value in biomedical research, making it a biosensor and important fluorescent probes in imaging assays. However, when quantum dots are used as fluorescent markers, there are still some problems, such as aggregation in solution, stability after biomarking, scintillation fluorescence, background fluorescence interference of complex biological samples, and potential cytotoxicity. and interference with cellular physiological processes.

The immunoassay technology based on phosphorescence technology is essentially a new type of time-resolved fluorescence technology, which not only improves the detection sensitivity and stability, but also can be combined with the instrument to quantify the target analyte due to the characteristics of its luminescent marker. and qualitative testing. Therefore, a time-resolved fluorescent biosensor based on phosphorescence technology is developed to interpret the results of detection chromatography test strips based on phosphorescence technology, so as to finally achieve rapid qualitative and quantitative detection of target analytes.

Contents of the invention

The purpose of the present invention is to solve the problem that the existing atomic fluorescence instruments are non-dispersive spectrometers. Due to the problem of spectral interference in the non-dispersive optical system, some elements cannot obtain accurate measurement results, and then provide a phosphorescence-based luminescence technology. Time-resolved fluorescent biosensors and their applications.

The purpose of the present invention is achieved through the following technical solutions:

A time-resolved fluorescent biosensor based on phosphorescence technology, comprising: an excitation optical path module, a photoelectric signal receiving and conversion module, a control system module, an amplification and shaping circuit, a decoding interface circuit and a data buffer module, the excitation optical path module includes a main detection An excitation light module and an auxiliary optical scanning module; the photoelectric signal receiving conversion module includes a main detection photoelectric conversion module and an auxiliary scanning photoelectric conversion module; the main detection excitation light module includes a first excitation light source, a first lens, a first filter sheet, a second lens, a beam splitter, a second filter, a third lens and a grating; the auxiliary optical scanning module includes a second excitation light source, a third filter, a fourth lens and a fifth lens; the The control system module includes a main circuit, a first motor, a second motor, a third motor, a mechanical transmission device, a display screen and a printing device; and the second lens into the detection area of the chromatography test strip, and the phosphorescent signal emitted by the detection area passes through the second filter, the third lens and the grating to the main detection photoelectric conversion module, and is input to the main circuit through photoelectric conversion and It is displayed on the display screen or printed by a printing device; the main circuit controls the second excitation light source to emit an excitation beam, which is emitted to the barcode area of the chromatography test strip through the third filter and the fourth lens, and the barcode area reflects the laser signal and then It is input to the main circuit through the fifth lens, auxiliary scanning photoelectric conversion module, amplification and shaping circuit, decoding interface circuit and data buffer module, and displayed on the display screen or printed by the printing device; the main circuit controls the excitation optical path module, photoelectric signal receiving and converting Movement of the module, the first motor, the second motor, the third motor and the mechanical transmission.

Application of a time-resolved fluorescent biosensor based on phosphorescence technology, the detection objects are pathogens, antigens, antibodies, drugs, hormones, drugs, Qualitative and quantitative detection of antibiotics, tumor marker target analytes, and pesticides, antibiotics, and additives in vegetables, fruits, meat and other foods and water sources.

The present invention has the following advantages: the present invention uses the phosphorescent luminescent material, that is, platinum/palladium porphyrin as the biomarker, and the result is displayed in the form of infrared light signal under the irradiation of green light, and can be interpreted by instruments, so as to realize the identification of the target Quantitative detection of test substances. The present invention divides the chromatographic test strips into sandwich method mode, competition method mode, indirect method mode and capture method mode according to the different ways of immune reaction of the test object, and different detection modes can be adopted according to the different properties of the detection objects Rapid and sensitive qualitative and quantitative detection and analysis of different analytes in samples.

Description of drawings

Figure 1 is a schematic diagram of the principle of phosphorescence generation;

Figure 2 is a standard working curve for the detection of the double-antibody sandwich method;

Fig. 3 is a standard working curve diagram of the competition method pattern detection;

4 is a schematic diagram of the chemical structural formula of the phosphorescent material;

5 is a schematic diagram of a time-resolved fluorescent biosensor based on phosphorescence technology;

Fig. 6 is a graph of excitation and emission spectra of phosphorescent materials.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings: the present embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation is provided, but the protection scope of the present invention is not limited to the following embodiments.

As shown in Figure 5, a time-resolved fluorescent biosensor based on phosphorescence technology involved in this embodiment includes: an excitation optical path module, a photoelectric signal receiving and converting module, a control system module, an amplification and shaping circuit 28, and a decoding interface circuit 29 and a data cache module 30, the excitation optical path module includes a main detection excitation light module 1 and an auxiliary optical scanning module 2; the photoelectric signal receiving conversion module includes a main detection photoelectric conversion module 3 and an auxiliary scanning photoelectric conversion module 4; The main detection excitation light module 1 includes a first excitation light source 14, a first lens 15, a first filter 16, a second lens 17, a beam splitter 18, a second filter 19, a third lens 20 and a grating 21; The auxiliary optical scanning module 2 includes a second excitation light source 22, a third filter 23, a fourth lens 24 and a fifth lens 25; the control system module includes a main circuit 7, a first motor 8, and a second motor 9 , a third motor 10, a mechanical transmission 11, a display screen 12 and a printing device 13; the light emitted by the first excitation light source 14 is emitted through the first lens 15, the first optical filter 16, the beam splitter 18 and the second lens 17 In the detection area 5 of the chromatography test strip 26, the phosphorescence signal sent by the detection area 5 is irradiated to the main detection photoelectric conversion module 3 through the second filter 19, the third lens 20 and the grating 21, and is input to the main detection photoelectric conversion module 3 through photoelectric conversion. The circuit 7 is also displayed on the display screen 12 or the printing device 13 prints; the main circuit 7 controls the second excitation light source 22 to emit an excitation beam, which is emitted to the chromatography test strip 26 through the third optical filter 23 and the fourth lens 24 The barcode area 6, the laser signal reflected by the barcode area 6 is then input to the main circuit 7 through the fifth lens 25, the auxiliary scanning photoelectric conversion module 4, the amplification and shaping circuit 28, the decoding interface circuit 29 and the data buffer module 30 and displayed on the display screen 12 The upper display or printing device 13 prints; the main circuit 7 controls the movement of the excitation optical path module, the photoelectric signal receiving conversion module, the first motor 8, the second motor 9, the third motor 10 and the mechanical transmission device 11, and the chromatography test strip 26 is arranged on the mechanical transmission device 11.

The phosphorescent light emission is the light emission of metalloporphyrin series fluorescent dyes, the excited light spectrum range of the metalloporphyrin is 390-420nm, and the emitted light wavelength range is 600-700nm.

The metalloporphyrins are platinum/palladium porphyrin compounds. The excitation wavelength of platinum porphyrin is 380nm, and the emission wavelength is 648nm; the excitation wavelength of palladium porphyrin is 393nm, and the emission wavelength is 667nm.

The display screen 12 is a touch liquid crystal screen.

The printing device 13 is an embedded thermal printing device.

Application of a time-resolved fluorescent biosensor based on phosphorescence technology, the detection objects are pathogens, antigens, antibodies, drugs, hormones, drugs, Target analytes such as antibiotics and tumor markers, as well as qualitative and quantitative detection of pesticides, antibiotics, additives, etc. in vegetables, fruits, meat and other foods and water sources.

In the invention, the standard curve of the standard substance to be measured and the phosphorescent signal intensity is established and stored in the chip of the IC card. For each determination, insert the IC card into the sensor of the present invention, and by detecting the phosphorescence signal intensity values of the test strip detection line and the quality control line, the analysis software can calculate the concentration value of the analyte from the standard curve and display it. Due to the uncontrollability of raw materials and production processes, the standard curve will also change, so each batch of test strips needs to be equipped with the necessary IC card.

For the quantitative detection items, the present invention realizes the quantitative detection by establishing the standard curve of the standard substance to be measured and the phosphorescence signal intensity. For qualitative testing items, the result judgment is realized by establishing the cut-off value of the analyte. The test result ≥ Cut-off value is positive, otherwise it is negative.

The sensor of the present invention is compact and light, easy to carry, and has a friendly operation interface. It only takes 3 minutes to complete a test, with only 20 seconds between each test, and 180 tests can be completed in one hour.

The working process of the sensor provided by the present invention mainly includes the following steps:

1. Insert the IC card into the sensor IC card socket.

2. The detection chromatography test strip that has completed the immune reaction is inserted into the detection card slot.

3. The optical system in the detection device starts, the light source converges the light through the external optical path, and projects it to the test strip detection area through the lens, optical filter, beam splitter, and lens successively, and the optical filter filters out the excitation of the required specific wavelength Stray light other than light.

4. The single excitation light emitted from the main detection excitation light path irradiates the detection area of the chromatography test strip, and the phosphorescent material on the detection area emits a phosphorescence signal under the action of the excitation light.

5. The phosphorescence signal emitted by the detection area is captured by the phosphorescence signal receiving and converting module of the sensor, which completes the conversion of the photoelectric signal. Receive the incident light, emit photoelectrons and multiply them to realize the amplification of photoelectron signals, and then convert the photoelectron signals into electrical signals by the solid-state detector, and output the electrical signals by the electrical signal reading and writing circuit, after being processed by the software analysis control system, The results are displayed on a digital display.

The detection objects of the present invention are target analytes such as pathogens, antigens, antibodies, drugs, hormones, drugs, antibiotics, tumor markers in whole blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces and prostatic fluid specimens, and Qualitative and quantitative detection of pesticides, antibiotics, additives, etc. in vegetables, fruits, meat and other foods and water sources.

In the present invention, for the quantitative detection items, the quantitative detection is realized by establishing a standard curve of the standard substance to be tested and the phosphorescence signal intensity. For qualitative testing items, the result judgment is realized by establishing the cut-off value of the analyte. The test result ≥ Cut-off value is positive, otherwise it is negative.

The present invention divides the chromatographic test strips into sandwich method mode, competition method mode, indirect method mode and capture method mode according to the different ways of immune reaction of the test object, and different detection modes can be adopted according to the different properties of the detection objects Rapid and sensitive qualitative and quantitative detection and analysis of different analytes in samples.

Sandwich test strips are mainly used to detect macromolecular proteins in samples, such as antigens and antibodies produced by pathogenic microorganisms. The method for detecting the antigen is the double-antibody sandwich method, and the method for detecting the antibody is the double-antigen sandwich method. In the preparation process of the double-antibody sandwich test strip, the phosphorescent material is firstly labeled with the specific antibody A of the target analyte (that is, it can only react with the epitope of the target analyte A), and fixed in the conjugate pad; The analyte-specific antibody B (that is, it can only react with the target analyte B epitope) is immobilized on the detection line of the analytical membrane; the secondary antibody that can react with the specific antibody A is immobilized on the quality control line of the analytical membrane. During the detection process, when both the detection line and the quality control line generate phosphorescence signals at the same time, it is a positive reaction result, indicating that the detection sample contains the target analyte; when the detection line does not generate phosphorescence signals but the quality control line produces phosphorescence signals, it is a positive reaction A negative reaction result indicates that the test sample does not contain the target analyte. In the sandwich method mode, the intensity of the phosphorescence signal of the detection line is directly proportional to the concentration of the target analyte in the sample, that is, the higher the concentration of the target analyte, the higher the intensity of the phosphorescence signal.

Replace the above-mentioned antibody A and antibody B with antigen A and antigen B to establish a double-antigen sandwich method test strip, which can detect the antibodies produced by pathogenic microorganism infection. The detection process and result judgment are the same as the double-antibody sandwich method for antigen detection.

The competition method test strip is mainly used to detect small molecule antigens and haptens in samples. Such as hepatitis B virus e antibody and core antibody, small molecule hormones, drugs, drugs, and pesticides and antibiotics left in food. In the preparation process of the competition test strip, firstly, the phosphorescent material is labeled with the specific antibody A of the target analyte (that is, it can only react with the epitope of the target analyte A), and is immobilized in the conjugate pad; then the target The antigen of the analyte (containing antibody A specific reaction antigen epitope A) is immobilized on the detection line of the analytical membrane; the secondary antibody that can react with the specific antibody A is immobilized on the quality control line of the analytical membrane. When the concentration of the target analyte in the sample is high, the detection line does not produce a phosphorescence signal but the quality control line produces a phosphorescence signal, which is a positive reaction result, indicating that the test sample contains a target analyte exceeding a certain concentration; All the control lines produce phosphorescence signal, which is a negative reaction result, indicating that the target analyte in the test sample is lower than a certain concentration or even zero. In the competitive method mode, the intensity of the phosphorescence signal of the detection line is inversely proportional to the concentration of the target analyte in the sample, that is, the higher the concentration of the target analyte, the lower the intensity of the phosphorescence signal.

Indirect method mode test strips are mainly used to detect IgG antibodies produced after pathogenic microorganism infection. In the preparation process of the indirect method test strip, first, the phosphorescent material is labeled with anti-antibodies (mainly anti-human immunoglobulin IgG antibodies) and fixed in the conjugate pad; then an antigen is fixed on the detection line of the analytical membrane; IgG was immobilized on the analytical membrane quality control line. During the detection process, when both the detection line and the quality control line generate phosphorescence signals at the same time, it is a positive reaction result, indicating that the detection sample contains the target analyte; when the detection line does not generate phosphorescence signals but the quality control line produces phosphorescence signals, it is a positive reaction A negative reaction result indicates that the test sample does not contain the target analyte. In the indirect method mode, the intensity of the phosphorescence signal of the detection line is directly proportional to the concentration of the target analyte in the sample, that is, the higher the concentration of the target analyte, the higher the intensity of the phosphorescence signal. The advantage of the indirect method is that as long as the detection line antigen is changed, the same phosphorescently labeled anti-antibody can be used to establish a method for detecting the corresponding antibody. Using staphylococcal protein A instead of anti-human immunoglobulin IgG antibody can realize the detection of IgG antibody in various animals. The indirect method mode is generally only suitable for detecting total antibody or IgG antibody. If the indirect method is used to directly measure the IgM antibody, because there is generally a relatively high concentration of IgG antibody in the sample, the latter will compete for the binding of the solid-phase antigen, so that a part of the IgM antibody cannot bind to the detection line of the analytical membrane, thereby affecting the detection sensitivity. . At the same time, rheumatoid factor will interfere with the detection of IgM, resulting in poor specificity.

The capture method mode test strip is mainly used to detect the IgM antibody produced after pathogenic microorganism infection. In the preparation process of the capture test strip, the phosphorescent material is firstly labeled with a certain antigen and fixed in the conjugate pad; then the anti-antibody (mainly anti-human immunoglobulin IgM antibody) is fixed on the detection line of the analytical membrane; The specific antibody that can react with the antigen is immobilized on the analytical membrane quality control line. During the detection process, when both the detection line and the quality control line generate phosphorescence signals at the same time, it is a positive reaction result, indicating that the detection sample contains the target analyte; when the detection line does not generate phosphorescence signals but the quality control line produces phosphorescence signals, it is a positive reaction A negative reaction result indicates that the test sample does not contain the target analyte. In the capture method mode, the intensity of the phosphorescence signal of the detection line is directly proportional to the concentration of the target analyte in the sample, that is, the higher the concentration of the target analyte, the higher the intensity of the phosphorescence signal.

The phosphorescently labeled biologically active molecules provided by the present invention include antigens, antibodies, anti-antibodies, staphylococcal protein A, receptor ligands, drugs, cells and the like.

The phosphorescent luminescent material that the present invention uses is platinum/palladium porphyrin compound, and structural formula sees accompanying drawing 4, any one or several groups in R1-R8 modifying group, can be amino (-NH2), carboxyl (-COOH), Sulfhydryl (-SH), isothiocyanate (-NCS), etc. are used to label biologically active molecules such as antigens and antibodies, and isothiocyanate (-NCS) is the first choice. .

Example 1: Quantitative detection of double-antibody sandwich method for detection of hepatitis B virus surface antigen HBsAg

1. Drawing of standard working curve:

First, the purified HBsAg standard was diluted with 1:10 normal human serum (diluted with pH7. 6 samples of ml, 50ng/ml, 100ng/ml, 200ng/ml. Secondly, each sample was tested 10 times with 10 HBsAg test strips, and the T value of the sample and the control C value read by the testing instrument for 10 times were respectively averaged, and finally the T value corresponding to each concentration was obtained according to the ratio of the two. /C results are listed in the table below. (Table 1)

Concentration (ng/ml) 0 10 25 50 100 200 T mean 0.227 210.974 447.395 879.403 1784.198 3770.301 C mean 13.833 12.983 13.766 13.264 12.873 13.037 T/C 0.02 16.25 32.5 66.30 138.61 289.2

The standard working curve was drawn with the T/C value as the X coordinate and the HBsAg concentration as the Y coordinate. The expression of the standard working curve after statistical fitting was: Y=0.6923X+1.5243, and the square of the fitting coefficient was R ² =0.9991. as shown in picture 2.

2. Actual test results:

The test strip of Example 1 was tested for performance, and the minimum detection limit was 0.01ng/ml. At the same time, clinical samples were tested. 58 cases of HBsAg clinical samples collected from the hospital (among them 37 were positive and 21 were negative) were simultaneously tested with colloidal gold immunochromatography test paper and this system in a double-blind method:

Colloidal gold immunochromatographic test paper method - 31 positive, 27 negative (ie 6 positive missed);

Platinum porphyrin test paper and instrument method - 37 were positive and 21 were negative, completely consistent with the actual results. At the same time, compared with the qualitative detection of colloidal gold immunochromatographic test paper, the platinum porphyrin test paper and instrument method gave the final accurate concentration of each sample.

These 58 cases of HBsAg clinical samples collected from the hospital were correlated with the HBsAg electrochemiluminescence chromatography test strip detection of Roche Company in the United States, and the electrochemiluminescence detection results were used as the X coordinates. Draw a correlation analysis curve with the result of the instrument method as the Y coordinate, the expression is Y=0.9993X-2.6157, and the correlation coefficient is r=0.9988. According to the statistical analysis, r>95%, P<0.01, there is a positive correlation.

In terms of intra-batch precision, the test strips of Example 1 were used to continuously detect at least 10 times for the samples whose contents were respectively high, middle and low, and calculate the coefficient of variation (CV). For HBsAg content of high value (100ng/ml), middle value (40ng/ml), low value (5ng/ml) samples were measured 10 times respectively, according to the data measured, using SPSS statistical method to analyze, to determine the results Mean ± standard deviation, high value 98.3±3.6ng/ml, CV2.9%; median value 39.6±1.8ng/ml, CV5.6%; low value 4.7±0.6ng/ml, CV7.9%; test results The CV values are all less than 15%.

In terms of precision between batches, utilize the test strip of embodiment 1, a HBsAg clinical positive sample is diluted 10 times with pH7. The coefficient of variation (CV) of repeated detection of this sample was calculated to be 4.73%. (Table 2)

Duplicate detection serial number 1 2 3 4 5 T/C 36.73 35.46 37.82 36.89 38.13 HBsAg concentration (ng/ml) 30.2 29.7 31.3 30.3 31.7 Duplicate detection serial number 6 7 8 9 10 T/C 35.48 38.24 37.63 34.77 34.82 HBsAg concentration (ng/ml) 29.7 31.8 31.2 29.1 29.2

It can be seen from the above detection that the detection method of the present invention has higher sensitivity and good repeatability while realizing accurate quantitative detection within and between batches.

Example 2: Qualitative detection of HIV antibodies by double-antigen sandwich method

1. Determination of the cut-off:

While measuring a large number of normal human serum samples, measure a considerable number of positive serum samples. If the measured value is normally distributed, then according to the characteristics of the μ test, the negative and positive cuts are firstly determined with a one-sided 99.5% confidence limit. -off value; if it is a non-normal distribution, the percentile method can be used to determine the cut-off value with one-sided 95% or 99%. After the cut-off value of the negative and positive population is determined, the cut-off value is determined according to the size of the "grey area" and considering the false positive and false negative rates in a comprehensive balance. The test result is positive if the measured value ≥ Cut-off value, otherwise it is negative.

2. Actual test results:

The test strip of Example 2 was tested for performance, and the minimum detection limit was 0.1 ng/ml. At the same time, clinical samples were tested. 65 cases of HIV clinical samples collected from the hospital (39 positive and 26 negative) were simultaneously tested with colloidal gold immunochromatography test paper and this system in a double-blind method:

Colloidal gold immunochromatographic test paper method - 36 positive, 29 negative (ie 3 positive missed);

Platinum porphyrin test paper and instrument method - 39 were positive, 26 were negative, completely consistent with the actual results. At the same time, compared with the qualitative detection of the colloidal gold immunochromatographic test paper, the platinum porphyrin test paper and the instrument method give the final concentration of each sample.

In terms of intra-batch precision, the test strips of Example 2 were used to continuously detect at least 10 times for the samples whose contents were respectively high, middle and low, and calculate the coefficient of variation (CV). For the HIV antibody content high value (40ng/ml), middle value (20ng/ml), low value (5ng/ml) each sample is measured 10 times, according to the data measured, adopt SPSS statistical method to analyze, to determine The results mean ± standard deviation, high value 38.3±3.2ng/ml, CV3.8%; median value 18.6±2.3ng/ml, CV6.3%; low value 4.3±0.8ng/ml, CV9.9%; detection The results showed that the CV values were all less than 15%.

In terms of precision between batches, utilize the test strip of embodiment 2, a HIV clinical positive sample is diluted 10 times with pH7. The coefficient of variation (CV) of repeated detection of this sample was calculated to be 6.26%. (table 3)

Duplicate detection serial number 1 2 3 4 5 T/C 21.80 20.86 19.10 21.76 20.28 HIV antibody concentration (ng/ml) 11.2 10.8 9.5 11.2 10.6 Duplicate detection serial number 6 7 8 9 10 T/C 19.81 19.58 20.58 22.73 19.13 HIV antibody concentration (ng/ml) 9.6 9.5 10.7 11.7 9.2

It can be seen from the above detection that the detection method of the present invention has higher sensitivity and good repeatability while realizing accurate quantitative detection within and between batches.

Embodiment 3: Quantitative detection competition method pattern detects morphine

1. Drawing of standard working curve:

First, dilute the pure morphine standard with pH7.20.02M PB buffer to prepare a series of concentration standards, the concentrations are: 0ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 400ng/ml, 800ng/ml 6 samples. Secondly, each sample was tested 10 times with 10 morphine test strips, and the T value of the sample detected by the 10 times of detection equipment and the control C value were respectively averaged, and finally the T value corresponding to each concentration was obtained according to the ratio of the two. /C results are shown in the table below. (Table 4)

Concentration (ng/ml) 0 50 100 200 400 800 T mean 1211.70 882.90 923.59 679.69 422.57 65.35 C mean 15.8 12.700 14.456 12.662 11.552 11.797 T/C 76.69 69.52 63.89 53.68 36.58 5.54

Take the T/C value as the X coordinate, and use the morphine concentration as the Y coordinate to draw a standard working curve. The expression of the standard working curve through statistical fitting is: Y=-11.406X+839.83, and the square of the fitting coefficient is R ² =0.993 . The results are shown in Figure 3: standard working curve for morphine detection.

2. Actual test results:

The test strip of embodiment 3 is tested for performance, and the minimum detection limit is 50ng/ml. At the same time, clinical samples were tested. 55 clinical samples (33 positive and 22 negative) of drug addicts collected from drug rehabilitation centers were tested in a double-blind method with colloidal gold immunochromatography test paper and this system at the same time:

Colloidal gold immunochromatography test paper method - 31 positive, 24 negative (ie 2 positive missed);

Platinum porphyrin test paper and instrument method - 33 were positive, 22 were negative, completely consistent with the actual results. At the same time, compared with the qualitative detection of the colloidal gold immunochromatographic test paper, the platinum porphyrin test paper and the instrument method give the final concentration of each sample.

In terms of intra-batch precision, the test strips of Example 3 were used to continuously detect at least 10 times for the samples whose contents were respectively high, middle and low, and calculate the coefficient of variation (CV). For the high value (800ng/ml), middle value (400ng/ml) and low value (100ng/ml) samples of morphine content, each sample was measured 10 times, and according to the data measured, the SPSS statistical method was used to analyze the results. Mean ± standard deviation, high value 786.8±22.7ng/ml, CV4.3%; median value 389.6±13.3ng/ml, CV6.2%; low value 102.1±7.9ng/ml, CV9.4%; test results The CV values are all less than 15%.

In terms of precision between batches, utilize the test strip of embodiment 3, a morphine clinical positive sample is diluted 10 times with pH7.20.02M PB damping fluid, detect at least 10 times continuously, the result is listed in the following table. The coefficient of variation (CV) of repeated detection of this sample was calculated to be 7.86%. (table 5)

Duplicate detection serial number 1 2 3 4 5 T/C 33.68 35.57 36.59 34.58 38.09 Morphine Concentration (ng/ml) 446.1 427.4 422.8 451.5 415.4 Duplicate detection serial number 6 7 8 9 10 T/C 35.22 37.56 36.88 34.65 35.82 Morphine Concentration (ng/ml) 428.5 421.6 429.2 449.4 426.5

It can be seen from the above detection that the detection method of the present invention has higher sensitivity and good repeatability while realizing accurate quantitative detection within and between batches.

Embodiment 4: Qualitative detection indirect method pattern detection hepatitis C virus IgG antibody

1. Determination of the cut-off:

While measuring a large number of normal human serum samples, measure a considerable number of positive serum samples. If the measured value is normally distributed, then according to the characteristics of the μ test, the negative and positive cuts are firstly determined with a one-sided 99.5% confidence limit. -off value; if it is a non-normal distribution, the percentile method can be used to determine the cut-off value with one-sided 95% or 99%. After the cut-off value of the negative and positive population is determined, the cut-off value is determined according to the size of the "grey area" and considering the false positive and false negative rates in a comprehensive balance. The test result is positive if the measured value ≥ Cut-off value, otherwise it is negative.

2. Actual test results:

The test strip of Example 4 was tested for performance, and the minimum detection limit was 0.2 ng/ml. At the same time, clinical samples were tested. 63 clinical samples of hepatitis patients collected from the hospital (among them 37 were HCV-IgG antibody positive, 26 were HCV-IgG antibody negative) were tested in a double-blind method with colloidal gold immunochromatography test paper and this system at the same time:

Colloidal gold immunochromatographic test paper method - 31 positive and 32 negative (ie 6 positive missed detection);

Platinum porphyrin test paper and instrument method - 37 were positive, 26 were negative, completely consistent with the actual results. At the same time, compared with the qualitative detection of the colloidal gold immunochromatographic test paper, the platinum porphyrin test paper and the instrument method give the final concentration of each sample.

In terms of intra-assay precision, the test strips of Example 4 were used to continuously detect at least 10 times for the samples whose contents were respectively high, middle and low, and calculate the coefficient of variation (CV). For HCV-IgG antibody content high value (40ng/ml), middle value (20ng/ml), each sample of low value (5ng/ml) measure 10 times respectively, according to the data of its determination, adopt SPSS statistical method to analyze, Expressed as the mean ± standard deviation of the measurement results, the high value is 40.8±3.7ng/ml, CV3.5%; the median value is 19.6±1.8ng/ml, CV5.2%; the low value is 5.1±0.7ng/ml, CV8.7% ; The CV values of the test results were all less than 15%.

In terms of precision between batches, using the test strip of Example 4, a clinically positive sample of hepatitis C patient was diluted 10 times with pH7.20.02M PB buffer solution, and at least 10 consecutive tests were performed, and the results are listed in the following table. The coefficient of variation (CV) of repeated detection of this sample was calculated to be 7.57%. (Table 6)

Duplicate detection serial number 1 2 3 4 5 T/C 36.22 34.07 38.06 35.46 38.51 HCV-IgG concentration (ng/ml) 10.7 9.5 11.6 10.1 12.1 Duplicate detection serial number 6 7 8 9 10 T/C 34.58 37.59 38.26 34.56 37.88 HCV-IgG concentration (ng/ml) 9.8 11.1 11.8 9.8 11.3

It can be seen from the above detection that the detection method of the present invention has higher sensitivity and good repeatability while realizing accurate quantitative detection within and between batches.

Embodiment 5: Qualitative detection capture method pattern detection hepatitis E virus IgM antibody (HEV-IgM)

1. Determination of the cut-off:

While measuring a large number of normal human serum samples, measure a considerable number of positive serum samples. If the measured value is normally distributed, then according to the characteristics of the μ test, the negative and positive cuts are firstly determined with a one-sided 99.5% confidence limit. -off value; if it is a non-normal distribution, the percentile method can be used to determine the cut-off value with one-sided 95% or 99%. After the cut-off value of the negative and positive population is determined, the cut-off value is determined according to the size of the "grey area" and considering the false positive and false negative rates in a comprehensive balance. The test result is positive if the measured value ≥ Cut-off value, otherwise it is negative.

2. Actual test results:

The test strip of Example 5 was tested for performance, and the minimum detection limit was 0.5 ng/ml. At the same time, clinical samples were tested. 58 clinical samples of hepatitis patients collected from the hospital (among them 35 were positive for HEV-IgM antibody, 23 were negative for HEV-IgM antibody) were used for double-blind detection with colloidal gold immunochromatography test paper and this system at the same time:

Colloidal gold immunochromatographic test paper method - 31 were positive, 27 were negative (that is, 4 positive missed detection);

Platinum porphyrin test paper and instrument method - 37 were positive and 21 were negative, completely consistent with the actual results. At the same time, compared with the qualitative detection of the colloidal gold immunochromatographic test paper, the platinum porphyrin test paper and the instrument method give the final concentration of each sample.

In terms of intra-batch precision, the test strips of Example 5 were used to continuously detect at least 10 times for samples whose contents were respectively high, medium and low, and calculate the coefficient of variation (CV). For HEV-IgM antibody content high value (50ng/ml), middle value (10ng/ml), low value (5ng/ml) each sample is measured 10 times respectively, according to the data of its determination, adopt SPSS statistical method to analyze, Expressed as the mean ± standard deviation of the measurement results, the high value is 50.8±3.7ng/ml, CV3.1%; the median value is 9.6±1.3ng/ml, CV5.8%; the low value is 4.6±0.8ng/ml, CV8.9% ; The CV values of the test results were all less than 15%.

In terms of batch-to-batch precision, using the test strip of Example 5, a clinically positive sample of a hepatitis E patient was diluted 10 times with pH7.20.02M PB buffer solution, and at least 10 consecutive tests were performed. The results are listed in the table below. The coefficient of variation (CV) of repeated detection of this sample was calculated to be 7.46%. (Table 7)

Duplicate detection serial number 1 2 3 4 5 T/C 46.54 44.72 43.57 46.48 48.22 HEV-I gM concentration (ng/ml) 5.6 4.6 29.1 5.5 6.8 Duplicate detection serial number 6 7 8 9 10 T/C 45.43 47.56 47.88 44.37 44.45 Concentration of HEV-IgM (ng/ml) 5.1 6.1 6.3 4.3 4.4

It can be seen from the above detection that the detection method of the present invention has higher sensitivity and good repeatability while realizing accurate quantitative detection within and between batches.

The above are only preferred specific implementations of the present invention. These specific implementations are all based on different implementations under the overall concept of the present invention, and the scope of protection of the present invention is not limited thereto. Anyone familiar with the technical field Within the technical scope disclosed in the present invention, any changes or substitutions that can be easily conceived by a skilled person shall fall within the protection scope of the present invention.

Claims (6)

1. A time-resolved fluorescent biosensor based on phosphorescent luminescence technology, characterized in that it includes: an excitation optical path module, a photoelectric signal receiving and converting module, a control system module, an amplification and shaping circuit, a decoding interface circuit and a data buffer module, the The excitation light path module includes a main detection excitation light module and an auxiliary optical scanning module; the photoelectric signal receiving conversion module includes a main detection photoelectric conversion module and an auxiliary scanning photoelectric conversion module; the main detection excitation light module includes a first excitation light source, a first lens, a first optical filter, a second lens, a beam splitter, a second optical filter, a third lens and a grating; the auxiliary optical scanning module includes a second excitation light source, a third optical filter, a fourth lens and a first Five lenses; the control system module includes a main circuit, a first motor, a second motor, a third motor, a mechanical transmission, a display screen and a printing device; the light emitted by the first excitation light source passes through the first lens, the first The optical filter, the spectroscope and the second lens are emitted into the detection area of the chromatography test strip, and the phosphorescent signal emitted by the detection area is transmitted to the main detection photoelectric conversion module through the second optical filter, the third lens and the grating, and then passed through the photoelectric signal. Convert the input to the main circuit and display it on the display screen or print on the printing device; the main circuit controls the second excitation light source to emit the excitation beam, which is emitted to the barcode area of the chromatography test strip through the third filter and the fourth lens. The laser signal reflected from the area is then input to the main circuit through the fifth lens, auxiliary scanning photoelectric conversion module, amplification and shaping circuit, decoding interface circuit and data buffer module, and displayed on the display screen or printed by the printing device; the main circuit controls the excitation light path The movement of the module, the photoelectric signal receiving conversion module, the first motor, the second motor, the third motor and the mechanical transmission device. 2. The time-resolved fluorescent biosensor based on phosphorescent luminescence technology according to claim 1, wherein the phosphorescent luminescence is metalloporphyrin series fluorescent dye luminescence, and the excitation light spectrum range of the metalloporphyrin is 390- 420nm, the emitted light wavelength range is 600-700nm. 3. The time-resolved fluorescent biosensor based on phosphorescence technology according to claim 2, wherein the metalloporphyrin is a platinum/palladium porphyrin compound. The excitation wavelength of platinum porphyrin is 380nm, and the emission wavelength is 648nm; the excitation wavelength of palladium porphyrin is 393nm, and the emission wavelength is 667nm. 4. The time-resolved fluorescent biosensor based on phosphorescence technology according to claim 1, wherein the display screen is a touch liquid crystal screen. 5 . The time-resolved fluorescent biosensor based on phosphorescence technology according to claim 1 , wherein the printing device is an embedded thermal printing device. 6. an application of the time-resolved fluorescent biosensor based on phosphorescent luminescence technology according to claim 1, wherein the detection object is in whole blood, blood plasma, serum, cerebrospinal fluid, urine, saliva, feces and prostatic fluid specimens Qualitative and quantitative detection of pathogens, antigens, antibodies, drugs, hormones, drugs, antibiotics, tumor markers, and pesticides, antibiotics, and additives in foods such as vegetables, fruits, meat, and water sources.

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